The use of inverted microscopes is ubiquitous in the life sciences and other fields that call for inspection of a sample in a liquid setting, in vitro. An inverted microscope views a sample through the bottom of a sample-containing vessel, such as a petri dish. The optical path includes an objective positioned beneath the vessel. To provide optimal optical properties, the sample vessel is constructed from a high-clarity material, usually glass, and the bottom of the vessel through which the sample is viewed is typically made as thin as practicable, oftentimes on the order of 170 micrometers. This is done for several reasons, first, a thin bottom of the vessel is made thin to allow the use of high numeric apertures in the inverted microscope's optics, which in turn have very short working distance, requiring the objective to be placed very close to the sample sitting at the bottom of the vessel. Also, a thin vessel bottom supports the use of fluorescence in observing the sample. Since glass naturally absorbs ultraviolet wavelengths, making the vessel bottom very thin facilitates improved transmission of these wavelengths from the sample to the objective.
Over the last 20 or so years, atomic force microscope (AFM) modules have been added to inverted microscopes to add AFM functionality. An AFM module is also referred to herein as an AFM head. An AFM is a type of microscope that “feels” a sample using a micro-fabricated probe tip mounted at the end of a cantilever microstructure. An AFM is able to view a sample at the nanoscale. The term nanoscale in the present context refers to a size of less than one micrometer. Modern-day high-performing AFMs can image and manipulate a sample with sub-nanometer resolution. With AFM technology, samples can be studied at the molecular, and sometimes even at the atomic, scale, which provides exceptional insight into such structures as cell membranes, DNA structures, and the like. Other relevant advantages provided by AFMs is their ability to measure the height of an object to produce a 3-dimensional image, to examine mechanical properties of the sample, and to manipulate the sample at the nanoscale.
In the operation of an AFM head, the probe tip is brought in close proximity to a surface of the sample such that the probe tip interacts with the sample through Van der Waals, capillary, electrostatic, and other forces that are significant at the nanoscale. The probe is scanned over the surface of the sample, while the deflection of the cantilever is observed and used as an input into a control system that continuously adjusts the height of the AFM probe to follow the topography of the sample. Scanning the sample with the AFM probe in this manner while recording the lateral coordinates (x, y), and the vertical coordinate (z) of the probe produces a three-dimensional image of the sample. An example of one such AFM is described in U.S. Pat. No. 6,057,546, the disclosure of which is incorporated by reference herein.
One long-felt problem experienced with the use of AFM heads in inverting microscopes in particular is the high-level of noise present in the AFM images. In the past, this problem has been addressed by slowing down the speed of the AFM scanning to operate the instrument in a stable regime. As such, the time needed to acquire an image of even a very small area of the sample has been on the order of 20 minutes or more. Such prolonged scan times severely limit the use of AFM heads, particularly for live samples such as bacteria or other microbes.
Another approach used to address the noise problem is eliminating the use of petri dishes altogether, using instead a smaller, slide-like vessel customized for the particular AFM instrument. This approach is not desirable because many users of inverted microscopes prefer the convenience of using standard petri dishes as the sample container.
A solution is therefore needed to improve AFM instruments, particularly their utilization in conjunction with inverted microscopes and petri dish sample vessels, to address the noise encountered in such applications.